Scaffold materials from glycosylated and PEGylated bovine serum albumin Kai Wang,1 Allan E. David,2 Young-Suk Choi,2 Yonnie Wu,3 Gisela Buschle-Diller1 1

Department of Polymer and Fiber Engineering, Auburn University, Auburn, Alabama 36849 Department of Chemical Engineering, Auburn University, Auburn, Alabama 36849 3 Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849 2

Received 10 November 2014; revised 6 January 2015; accepted 4 February 2015 Published online 26 February 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35430 Abstract: Bovine serum albumin has been PEGylated and glycosylated to create mimetic materials for the extracellular matrix (ECM) with potential tissue engineering applications. Different surfaces for cell adhesion were achieved by crosslinking the initial albumin product and forming either a coating or a sponge-like three-dimensional morphology to mimic the mesh structure of natural ECM. The biocompatibility of the albumin matrix with mammalian cells was evaluated using cell culture assays with NIH 3T3 cells. The results indicated that glycoprotein composition and specific morphology

of the assembly can improve the cell growth environment. These ECM mimetic structures might eventually be considered to serve as alternatives for the more expensive collagen and elastin based ECM substances currently in use in tissue C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part engineering. V A: 103A: 2839–2846, 2015.

Key Words: BSA, glycosylation, biomaterials, cell adhesion, biocompatibility

How to cite this article: Wang K, David AE, Choi Y-S, Wu Y, Buschle-Diller G. 2015. Scaffold materials from glycosylated and PEGylated bovine serum albumin. J Biomed Mater Res Part A 2015:103A:2839–2846.

INTRODUCTION

Artificial scaffold materials that can mimic the extracellular matrix (ECM) are the future material resources in tissue engineering. They are required to provide a functional network for appropriate cell interactions and stabilize newly developed tissue, while serving as a nurturing reservoir for molecules such as growth factors.1–4 In order to achieve these goals, artificial ECM must be designed to be biocompatible, nontoxic, nonimmunogenic, biodegradable, and highly porous but at the same time show adequate mechanical properties.2–4 Numerous materials have been investigated to obtain artificial ECM. Natural biopolymers, such as collagen, hyaluronic acid, fibronectin, chitosan, alginate, and silk, could be expected to be well accepted as scaffolds in physiological environments.4–7 Compared with synthetic polymer scaffolds, naturally derived ECM components containing polysaccharides have the advantage of good cell adhesion, desired mechanical properties, minimized foreign body reaction, and lower risk of causing inflammation.5,8 In a number of studies, artificial ECM has been created by incorporating natural ECM proteins to enhance cell adhesion.6,9 Proteins with RGD sequence, such as collagen and fibronectin, showed cell adhesion via attachment of integrin.7 Collagen (type II), in a sponge shape with proteoglycans (hyaluronic acid or chondroitin sulfate), has been reported to form well-constructed ECM in vitro or in vivo

with chondrocytes.10,11 Fibroin and hyaluronic acid, as well as type I collagen, might be used in bone tissue engineering.12 Together with the bone morphogenetic protein-2, these materials can provide a biomimetic environment that facilitates the growth of new bone from stem/progenitor cells.13 However, the cost of high-performance artificial ECM materials, like those based on collagen, hyaluronic acid, or fibronectin, can be high.14 Therefore, efforts have been made to find cost-effective ECM materials with enhanced functions. Like natural polymers, synthetic materials were grafted with RGD or ECM protein mimicking peptides, to enhance cell adhesion.14–16 The RGD sequence grafted onto polylactic acid films has achieved rapid adhesion of osteogenic precursor cells and proliferated cell growth.16 RGD was also grafted as pendant in a polyethylene glycol (PEG)-based hydrogel to encapsulate human mesenchymal stem cells.17 The cost for these hybrids with synthetic polymers is comparatively low. The stability of these peptides, however, was found inadequate, and the products were susceptible to degradation.14–16,18 Cyclic structures of the peptides were introduced to improve the stability. Because of low reaction yields, the application of those peptides is limited.18 In other research, mono-, oligo-, and polysaccharides (e.g., hyaluronic acid, alginate, or galactose) were employed as ligands or grafts for tissue engineering materials. It was

Correspondence to: G. Buschle-Diller; e-mail: [email protected]

C 2015 WILEY PERIODICALS, INC. V

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FIGURE 1. Synthesis of glycosylated and PEGylated BSA (GA: gluconic acid and LA: lactobionic acid).

found that galactose may serve as asialoglycoprotein receptor for hepatocytes.19,20 Alginate combined with galactosylated chitosan may have easily formed stable hydrogels with improved cell adhesion.21 Although some of the ECM mimics that contained polysaccharide components are still expensive, the cost of polysaccharide and galactosylated polymers is significantly lower than that of purely protein-based materials. PEG is a biocompatible polymer and has been applied for modification of numerous synthetic and natural polymers4 to introduce thermo-responsiveness in cell adhesion materials.21 These studies could lead to effective alternatives for tissue engineering materials. Bovine serum albumin (BSA) is an abundant animal protein resource, which has not yet been fully utilized. Limited research on BSA nanoparticles, preformed hydrogels, and fibers were conducted by different researchers as drugs and/or bioactive molecular delivery vehicles.22,23 Unlike collagen or fibronectin, native BSA does not have the b1 integrin receptor (RGD sequence).24 Research of Kawamura et al.25 showed that a cell-adherent BSA-based membrane could be created for some nonadhesive cells. However, the function of this material was designed for cell anchoring and transferring, but not proliferation or differentiation of cells. To our knowledge, BSA has not been used as the scaffold material for tissue engineering in vivo or in vitro. Incorporating ECM-simulating components into BSA could combine the merits of these components with affordable cost of the BSA-based material. In this study, BSA is grafted with a monosaccharide attached to PEG chains on specific sites to mimic glycoprotein and glycosaminoglycan in natural ECM, as a model for artificial ECM that mimics the chemical components and structure of natural ECM. BSA was reduced to obtain a linear structure and crosslinked to form a mesh-like morphology. Cell adhesion was tested on the BSA network and compared with that of other scaffold materials. The result indicated that the material may possess advantageous prop-

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erties over collagen-, fibronectin-, or polypeptide-based ones. MATERIALS AND METHODS

Materials BSA, collagen type I from bovine calf skin, tetramethylethylenediamine, ethylenediaminetetraacetate (EDTA), and lactobionic acid (LA, 4-O-b-D-galactopyranosyl-D-gluconic acid) were obtained from Sigma-Aldrich (St. Louis, MO); PEG diamine (PEGDA, molecular weight 200 g/mol), dithiothreitol (DTT), 1-ethyl-3-(3-dimethlyaminopropyl)carbodiimide (EDC), and gluconic acid (GA) were obtained from Alfa Aesar (Ward Hill, MA). Trypsin (1:250, tissue culture grade) and phosphate-buffered saline were purchased from Amresco (Solon, OH), and hydrogen chloride was obtained from BDH (Poole, UK). Synthesis The schematic of the BSA modification is shown in Figure 1. Briefly, 0.2 g (3 mmol) BSA was dissolved in 50 mL of 0.05M tetramethylethylenediamine solution and allowed to react with 0.023 g (0.15 mmol) DTT for 12 h (pH 8) at room temperature.20 Subsequently, 0.3 g (0.15 mmol) PEGDA was added, the pH of the solution adjusted to 4.5 with hydrochloric acid, and the reaction was continued for another 12 h. Finally, 0.5 g sugar acid (SA, including GA or LA) and 0.05 g EDC were added and allowed to react for 24 h. The product was separated and purified multiple times by centrifugal filtration (molecular weight cutoff 30 kDa) with deionized water as the eluent. The concentration of the final product was determined by UV-vis spectroscopy.24 Coating and lyophilization The coating solution consisted of 0.5 mL purified BSAPEGDA-SAs aqueous solution (1%, 2%, and 4% w/v, determined by UV-vis spectroscopy) mixed with 4.5 mL deionized water and dispersed onto 85 3 13 mm plastic petri dishes

GLYCOSYLATED AND PEGYLATED BSA SCAFFOLDS

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(VWR, Radnor, PA; nontreated polyethylene). Coating was achieved by removing solvent in a desiccator overnight at room temperature. EDC (0.05 mL, 1% (w/v)) in a methanol:water (9:1) solution was pipetted onto the coating, and the samples were dried in a desiccator at room temperature. A sponge-like morphology can be obtained by lyophilization (samples are termed “3D”). To prepare samples with 3D topology, 0.5 mL of purified BSA-PEGDA-SA aqueous solution (1%, 2%, and 4% w/v, determined by UV-vis spectroscopy) was added to each well on a 24-well plate. Samples were prepared by adding EDC to each well in increasing amounts (10, 20, 50, 100, 200, and 500 mg, respectively). The plate was frozen at 218  C overnight and lyophilized under 5 mTorr vacuum for 24 h. The dried product was immersed in 0.01% (w/v) EDC solution (methanol:water 9:1, pH 4.5) for 24 h. EDC solution was carefully pipetted out and rinsed with solvent (methanol:water 9:1). The product was frozen and lyophilized using the previous conditions. Characterization The samples were characterized by proton nuclear magnetic resonance (1H-NMR) spectroscopy using a Bruker 400 MHz spectrometer. Samples were first dried in a desiccator under vacuum and dissolved in 0.5 mL deuterium oxide solvent. The molecular weights of modified BSA were measured by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF; Bruker Microflex LT MALDI-TOF mass spectrometer with the FlexControl software). The mass range was detected from 20 to 200 kDa, with sinapic acid as the matrix. As described in a previous work,26 UV-vis spectroscopy was used (Thermo Scientific Genesys 6 spectrometer; Thermo Scientific, Waltham, MA; quartz cuvettes with 10 mm path length) to measure the yield and concentration of the BSA moiety before the coating of the samples. The absorption coefficient for modified BSA used was 3,443,824 M21 cm21 (280 nm). The morphology of the coated samples was studied with a Carl Zeiss EVO 50 scanning electron microscope at 20 kV scanning voltage. Gold coating was applied by a sputter coater prior to imaging. Circular dichroism (CD) spectra were generated with a JASCO-810 spectropolarimeter (Jasco, Tokyo, Japan) within the wavelength ranges from 180 to 300 nm. Quartz cuvettes with 2 mm path length were used, and the average molecular weight of BSA residues used was 114 g/mol. Samples were dissolved in 0.05M phosphate buffer at pH 7.4 with 1 mg/mL protein. Protein moiety ellipticity was determined by Eq. (1)24,25: u ¼ 114uk =10dc

(1)

where hk is the observed ellipticity, d is the light path, and c is the concentration.

(Thermo Scientific) with 10% fetal bovine serum (Thermo Scientific), 1% antibiotic–antimycotic (10,000 units/mL of penicillin, 10,000 mg/mL of streptomycin, and 25 mg/mL of Fungizone, Gibco, Life Technologies, Grand Island, NY), in 100 3 20 mm cell culture petri dishes (Corning Costar, Corning, NY; tissue-culture treated polystyrene (TPS)). Cells were incubated in 5% CO2 at 37  C. Culture medium was changed every 72 h. Cells were detached with 1 mL trypsinEDTA solution (0.25% trypsin and 0.02% EDTA), and the suspension was centrifuged at 1500 rpm for 5 min. Cells were diluted in 2 mL culture medium and counted using a hemocytometer. Cell seeding and viability assay Harvested NIH 3T3 cells were seeded 5 3 103 cells per well onto a 24-well plate (Corning Costar) of coated BSA-PEGDAGA/LA and uncoated and collagen coated controls; six wells per type. Then, 0.5 mL BSA-PEGDA-GA/LA solution with 4 mg/mL concentration was coated onto each well, and the samples were dried under vacuum; 0.05 mL 1% (wt %) EDC solution (methanol:water, 9:1) was added to the surface and rinsed with methanol-water solution (9:1), then dried under vacuum. Collagen-coated wells used 0.5 mL collagen (1 mg/ mL 0.02M acetic acid solution). For the 3D structure, the preparation followed the same procedure as described earlier, except that a 4% solution of BSA-PEGDA-SA containing 100 mg EDC was used. Plates were sterilized by longwavelength UV light for 2 h prior to cell seeding. The cells were incubated for 4, 10, and 20 days in the case of coated samples, and 1, 3, and 7 days in the case of samples with 3D structure. Cells were examined under optical and inverted phase microscopes; cells were fixed with 3% aqueous glutaraldehyde solution before imaging. Cells cultured on TPS were used as the control group. The viability of cells was examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Biotium). Cells in each well were incubated with 0.05 mL MTT assay reagent with 0.5 mL full culture medium for 2 h. The culture medium was withdrawn and then 1.5 mL DMSO was added. The resulting solution was transferred to a well in a 96-well plate, and the absorption was examined by spectrophotometer at wavelengths of 570 and 630 nm (residual media absorption). The cell viability was determined by Eq. (2). Cell viability ¼ ðOD570nm ðSampleÞ2OD630nm ðSampleÞÞ= ðOD570nm ðControlÞ2OD630nm ðControlÞÞ

(2)

OD is the spectrophotometric reading of each well at the selected wavelength. The average values of six samples in each group were normalized, and the significance level was determined by the Student’s t-test. RESULTS

Cell culture NIH 3T3 cells (ATCC CRL-1658) were cultured in 4.5 g/L glucose-containing Dulbecco’s modified Eagle’s medium

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | SEP 2015 VOL 103A, ISSUE 9

Synthesis Figure 2 shows a schematic overview of the procedure to create BSA-based ECM mimics. DTT reduced the disulfide

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FIGURE 2. Production of BSA-based ECM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

bonds of BSA at alkaline pH. As a consequence of decreasing the pH, the globular structure of BSA began to unfold and transform into an extended form.25 Grafting of the PEG

chain occurred via the reaction of ANH2 groups on PEGDA and acidic functional groups of the protein, creating a long hydrophilic chain that could mimic the glycosaminoglycan component in the glycoprotein receptors.20 Finally, BSA molecules were intermolecularly crosslinked between ACOOH and ANH2 groups, and a glycosylated BSA mesh structure was created, which mimics the molecular structure of natural ECM (Fig. 2). The yield of BSA-PEGDA-GA was 67% (4% w/v solution, 3.5 mL), and the yield of BSA-PEGDA-LA was 59% (4% w/v solution, 3.1 mL). The BSA product was analyzed by 1H-NMR spectroscopy (Fig. 3). All the BSA-PEGDA-SAs showed characteristic peaks for both PEG and saccharide. In Figure 3(a), AOH groups of grafted saccharide acid shifted at d 5 3.5–4.1 ppm for GA and d 5 3.5–4.5 ppm for LA. The d 5 3.1–3.3 ppm peaks represent the ethoxyl groups, and d 5 2.6–2.8 ppm peaks represent the ANH groups bound to PEGDA. As shown in Figure 3(b,c), by the 2D correlation spectroscopy (COSY) method, the diagonal signals were clearly present. The cross signal of spots at d 5 2.6 ppm and d 5 4.0 ppm were from ANH2 and saccharides, respectively, indicating the bond formation between BSA-PEGDA and saccharide acid grafted via ANH groups of PEGDA.

FIGURE 3. (a) 1H-NMR spectra of BSA-PEGDA-GA and BSA-PEGDA-LA. (b) 2D COSY 1H-NMR spectra of BSA-PEGDA-GA. (c) 2D COSY 1H-NMR spectra of BSA-PEGDA-LA. All spectra were obtained with deuterium oxide at 400 MHz.

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FIGURE 4. CD spectra of BSA-PEGDA-GA and BSA-PEGDA-LA in 50 mM phosphate-buffered saline, pH 5 7.4. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

The molecular weights of BSA-PEGDA-SAs were measured by MALDI-TOF. Compared with 66.5 kDa for BSA, the molecular weight of BSA-PEGDA-GA was at 78.4 6 0.5 kDa and that of BSA-PEGDA-LA was 78.1 6 0.4 kDa. Thus, the grafting ratio of the BSA-PEGDA to GA was approximately 1:30 and that of BSA-PEGDA to LA was about 1:20. There was no indication of the presence of crosslinks or oligomers of BSA resulting from PEGylation and glycosylation reactions other than that from the crosslinking by EDC. The CD spectra of BSA-PEGDA-SAs were compared with that of native BSA (Fig. 4). A significant reduction in ahelix amount, about two-third compared with that in native BSA (absorption at 210 nm and 225 nm), clearly indicated that these molecules have unfolded under the reaction conditions (low pH) and provided an extended conformation.

Coating and aggregation BSA-PEGDA-SAs samples were deposited onto polyethylene petri dishes and dehydrated under vacuum. Different morphologies were formed at different starting concentrations as shown in Figure 5(a–c). At 1% (w/v), a dense structure of aligned fiber-like fragments was observed. It is possible that the aggregation at low concentration did not occur in a precise manner. When the concentration was increased to 2%, fine fibers were developed after dehydration.23 Finally, with a concentration at or above 4%, a thin film appeared on the petri dish surface. It seems that a concentration of at least 4% is required to coat the area if a concise consistency of the coating is desired. The cross-sections of the assembly revealed that the coating was

Scaffold materials from glycosylated and PEGylated bovine serum albumin.

Bovine serum albumin has been PEGylated and glycosylated to create mimetic materials for the extracellular matrix (ECM) with potential tissue engineer...
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